A selective and atom-economic rearrangement of uridine by cascade biocatalysis for production of pseudouridine

As a crucial factor of their therapeutic efficacy, the currently marketed mRNA vaccines feature uniform substitution of uridine (U) by the corresponding C-nucleoside, pseudouridine (Ψ), in 1-N-methylated form. Synthetic supply of the mRNA building block (1-N-Me-Ψ−5’-triphosphate) involves expedient access to Ψ as the principal challenge. Here, we show selective and atom-economic 1N-5C rearrangement of β-d-ribosyl on uracil to obtain Ψ from unprotected U in quantitative yield. One-pot cascade transformation of U in four enzyme-catalyzed steps, via d-ribose (Rib)-1-phosphate, Rib-5-phosphate (Rib5P) and Ψ-5’-phosphate (ΨMP), gives Ψ. Coordinated function of the coupled enzymes in the overall rearrangement necessitates specific release of phosphate from the ΨMP, but not from the intermediary ribose phosphates. Discovery of Yjjg as ΨMP-specific phosphatase enables internally controlled regeneration of phosphate as catalytic reagent. With driving force provided from the net N-C rearrangement, the optimized U reaction yields a supersaturated product solution (∼250 g/L) from which the pure Ψ crystallizes (90% recovery). Scale up to 25 g isolated product at enzyme turnovers of ∼105 mol/mol demonstrates a robust process technology, promising for Ψ production. Our study identifies a multistep rearrangement reaction, realized by cascade biocatalysis, for C-nucleoside synthesis in high efficiency.

We therefore here focused on the Ψ-5′-monophosphate (ΨMP) Cglycosidase YeiN 68,69,73,74 . The enzyme catalyzes naturally the hydrolysis of ΨMP into uracil (Ura) and D-ribose 5-phosphate (Rib5P) 69 . The reverse YeiN reaction was promising for Ψ synthesis because of two characteristics in particular 73 . Unlikely for a nominal hydrolysis performed in water, the reaction equilibrium lies far on the ΨMP side; and YeiN provides absolute β-stereo-direction of the C-ribosylation of uracil 69,73 . Reaction of the isolated ΨMP with an unspecific phosphatase gives Ψ 73,74 . Asking whether the YeiN reaction could become suitable for Ψ production in principle, we immediately noticed the fundamental need for reaction integration through an innovative (cascade) reaction concept. The chemically intriguing N-C rearrangement by Ψ synthase on RNA-bound U drew our attention. This led to the idea of a multienzyme cascade transformation, with C-glycosylation as the central step, to achieve the same net conversion of the free U. The U is an expedient starting material available in bulk quantities by large-scale fermentation [75][76][77] . Inspired by the natural salvage pathways of pyrimidine nucleosides, we here combine the YeiN (EC 4.2.1.70) reaction with the reactions of U phosphorylase (EC 2.4.2.3) and phosphopentomutase (EC 5.4.2.7) to promote a one-pot transformation of U into ΨMP (Fig. 2).
The further addition of a ΨMP-specific phosphatase (EC 3.1.3.5), discovered in this study for the purpose of controlled recycling of free phosphate, enables the U rearrangement into Ψ (Fig. 2). The U transformations into ΨMP and Ψ are both diastereoselective and atomeconomic. Driven by the β-C-glycosylation, they proceed in quantitative yield to final concentrations at the product solubility limit and with a productivity of ∼40 g/L/h. Performed at a scale of 25 g isolated Ψ, the enzyme cascade reaction is demonstrated as a robust biocatalytic process technology that is highly promising for use in production.

Design of the multienzyme cascade reaction
Domino reaction in three or four enzymatic steps was designed for U conversion into ΨMP or Ψ, respectively (Fig. 2). As shown in Table 1, the phosphorolysis of U involves only a limited driving force to give Rib1P and uracil, but the isomerization of Rib1P into Rib5P advances thermodynamically downhill. There is additional pull from the C-C coupling so that the overall conversion of U into ΨMP should readily proceed. The ΨMP hydrolysis is largely irreversible. The chemical reactions do not involve an immediate proton uptake or release. Phosphate group protonation does not change over the different steps (Rib1P, Rib5P, ΨMP). pH independence of the overall reaction equilibrium is thus implied, eliminating the pH as a factor of the chemical transformation. All reactants are stable under mild reaction conditions in water. Reactant solubility is discussed later.
Given the requirement of up to four enzymatic reactions telescoped in one pot, we considered enzyme compatibility regarding dependence of their activity on the bulk conditions (e.g., pH, temperature, ionic strength). The YeiN used is from E. coli and so we selected the already known other enzymes (U phosphorylase, UP; phosphopentomutase, DeoB) also from E. coli. A phosphatase suitable for use in the cascade reaction needed to be discovered. As described in a separate section below, we focused our search on the diversity of  29,30 or the reconstruction of the uracil nucleobase on a ribofuranosyl precursor 25 . E. coli phosphatases and identified the enzyme Yjjg. Good functional overexpression in E. coli was an additional point considered and it was confirmed by the experiments (Table 1, Supplementary Fig. 5). All enzymes were isolated by His-tag chromatography and exhibited specific activities (Table 1) within the range given in the BRENDA database 78 . A holistic approach of multienzyme system characterization (see later) was used to identify operational conditions of pH (7.0) and temperature (30 or 40°C) for synthesis performed in one-pot transformations. Kinetic parameters of the individual enzymes were determined under these conditions ( Supplementary Fig. 1) and are summarized in Table 1. The substrate K M values are all in the low mM range or even smaller. The substrate concentrations used, and the intermediate concentrations accumulating, in the cascade transformations exceed the corresponding K M values by several order of magnitude. A high ratio of substrate concentration/K M is, in general, beneficial for an efficient reaction into product. The DeoB is known to be somewhat vulnerable to inhibition by phosphate 79 . We determined the phosphate inhibition against Rib1P and showed it to be noncompetitive with a K i of 0.6 mM ( Supplementary Fig. 2, Table 1). The inhibition by phosphate appears to be partial: DeoB retains about 10% of its basal activity in the absence of phosphate under the highphosphate (≤ 1.0 M) conditions used for the cascade transformations (Table 1). Stability parameters were also obtained for the activity as well as for the protein structure (Table 1). UP is the most stable among the four enzymes used. It features a remarkable stabilization of structure by 1.0 M phosphate, reflected by 17°C increase in melting temperature compared to 0.1 M phosphate (Table 1; Supplementary  Fig. 3). YeiN and Yjjg exhibit melting temperature (T m ) in the range 36-46°C and lose activity gradually (∼50% in 16 h) even at 30°C (Supplementary Figs. 3,4). DeoB exhibits a T m larger considerably (T m ≥ 11°C) than the T m of YeiN and Yjjg (Table 1). Its activity is however not as resistant as one might expect from the T m . The DeoB is inactivated at a rate comparable to the inactivation rates of YeiN (Supplementary Fig. 4). Yjjg is the least stable enzyme.

Three-step conversion of U into ΨMP
The cascade reaction is shown in Fig. 3a. Offering U (244 g/L; 1.0 M) with phosphate present in 1.5-fold molar excess, the conversion into ΨMP proceeds to completion (≥95%), as shown in Fig. 3b. The evidence constitutes an excellent demonstration of the feasibility of the proposed three-enzyme cascade in Fig. 3a. The overall transformation happens in two kinetic phases, based on fast release of free Ura from U and slower consumption of uracil by β-C-ribosylation with Rib5P (Fig. 3b). Accumulation of the intermediary Ura to a concentration of ∼450 mM is worth emphasizing because it reflects a temporary supersaturation of the reaction mixture in Ura, exceeding the Ura solubility limit in water by ∼20-fold (Supplementary Table 1). In the isolated YeiN reaction, ∼25 mM Ura are maximally soluble (Supplementary Fig. 6). The curious delay in nucleation of the released Ura brings about a totally unanticipated feature of the cascade reaction that can have considerable advantage for the process engineering: tight monitoring of the enzyme activity ratio is not necessary to maintain control over the Ura solubility during the transformation. Were the formation of insoluble Ura happening at a rate comparable to the ΨMP formation rate, the overall reaction rate would have to be strictly limited by the rate of U conversion into Ura and Rib1P. Requirement to tune the flux through the individual steps of the domino reaction precisely would render even the basic reaction optimization a complicated task 80,81 .
In Fig. 3b, given the specific activity of the mutase DeoB considerably lower than that of UP and YeiN, the isomerization of Rib1P to Rib5P was rate-limiting overall ( Supplementary Fig. 7). DeoB is additionally inhibited by phosphate, as already mentioned (Table 1), plausibly explaining the ΨMP formation rate (∼2 mM/min) about 30-fold lower than the nominal DeoB activity used in the reaction at 30°C with 1.5 M phosphate.
Encouraged by these initial results, we systematically explored major reaction parameters with the aim of delineating the boundaries of the ΨMP synthesis. The ΨMP formation rate is largely independent of the pH in the range 6.5-7.5, but drops at pH 8.0 (       Fig. 11). This provides a simple scaling factor of reaction engineering for facile process optimization towards conversion efficiency and costeffectiveness.
Lastly, we consider the mechanistic requirement of DeoB to become activated via phosphorylation. The reversible conversion of Rib1P into Rib5P involves intramolecular phosphoryl transfer via a covalent DeoB phosphoenzyme intermediate. As in other sugar phosphate mutases, the activated DeoB state is that of enzyme phosphorylated at the active site nucleophile (Thr98 of DeoB). Sugar diphosphates, such as Rib-1,5-diphosphate (Rib1,5diP) and D-glucose-1,6-diphosphate (Glc1,6diP), serve as catalytic activators required in low micromolar concentration 82 . We show that the ΨMP formation rate is the same irrespective of whether Glc1,6diP (a costly reagent) is supplemented ( Supplementary Fig. 12), indicating that activator addition is not necessary. A plausible explanation of the result is that the DeoB as-isolated is in the activated phosphoenzyme state. We note however that phosphoenzyme decomposition (e.g., by hydrolysis) could be a relevant factor of stability of the DeoB activity. Evidence of activity loss at temperatures of 20°C below the T m suggests that inactivation might be caused by events different from thermal denaturation. At this stage of the development, however, enzyme stability was not an issue and its further exploration was left for consideration in the future.
Gram-scale preparative synthesis of ΨMP is performed in 5.0 mL volume, reflecting a scale-up factor of 50 compared to the reactions used in development. The reaction time courses obtained at the different scales are superimposable, as shown in Supplementary Fig. 13.
The excellent efficiency of the enzymatic transformation comes with the important benefit of a greatly simplified product isolation in technical-grade purity. Enzymes are removed by ultrafiltration, enabling their recycling, and the reaction mixture is lyophilized. The solid product recovered in ≥ 95% yield (based on UV-absorbance at 260 nm) is ΨMP of ≥97% HPLC purity (NMR, phosphate assay; Supplementary Figs. 14-20), with uracil, phosphate and potassium as minor impurities (∼1% by weight each) and traces of Mn 2+ present.
We made an effort to demonstrate the usability of the synthetic ΨMP in a follow-up transformation into a compound (ΨTP) involving more immediate applied interest than ΨMP itself. The ΨTP is substrate of in vitro transcription for mRNA synthesis. We use enzyme cascade phosphorylation of ΨMP (20 mM; 10 mL scale) according to protocol of our earlier study 73 (Supplementary Fig. 21a). The ΨMP conversion proceeds to 90% yield ( Supplementary Fig. 21b), giving a final ΨTP concentration of 18 mM (9.7 g/L). The product is isolated by anion exchange chromatography followed by lyophilization to obtain a white crystalline ammonium salt of ΨTP (∼100 mg). The chemical structure of the ΨTP is verified by 1 H, 13 C, and 31 P-NMR (Supplementary Figs. [22][23][24] and the purity is shown as ≥95% (Supplementary Fig. 25). These results validate the as-isolated ΨMP in technical-grade purity as fully sufficient for ΨTP synthesis.

Discovery of Yjjg as a ΨMP-specific phosphatase
The three-step rearrangement of U appears to be well-suitable for ΨMP production. However, we figured that integration with existing chemical routes of RNA building block synthesis is better established for Ψ than ΨMP. Domino reaction in four steps is therefore designed to obtain Ψ from U (Fig. 2). In this reaction, phosphate serves to shuttle the Rib moiety and is recycled in the last step of the reaction. Proper function of the coupled enzyme system for the proposed synthesis relies on phosphate release specifically from ΨMP. A phosphatase well active with ΨMP and able to discriminate against Rib1P and Rib5P is not known. Commercial enzymes (e.g., calf intestine phosphatase) are not usable due to lack of specificity 83 . In a genome-wide study of E. coli phosphatases, Kuznetsova et al. analyzed the substrate spectrum of 23 enzymes of the structural superfamily of haloalkane dehalogenases. The phosphatase Yjjg (oher name: HAD5) exhibited fast turnover (k cat ≥ 16 s −1 ) with pyrimidine monophosphates (UMP, dTMP, TMP)  whereas no activity with Rib5P was detected [84][85][86] . We show here that in terms of catalytic efficiency (k cat /K M ) the Yjjg prefers ΨMP (4.1 mM −1 s −1 ) about 500-fold to Rib5P (0.008 mM −1 s −1 ) as a substrate for dephosphorylation ( Supplementary Fig. 1f,g). This particular enzyme specificity thus renders Yjjg a clear candidate phosphatase to be used in the synthesis of Ψ from U.

Four-step conversion of U into Ψ
We performed the cascade reaction as before for ΨMP production, with the exception that Yjjg is included and phosphate is added to just 10 mol% of the U substrate (four-enzyme cascade, Fig. 2). Pleasingly, as shown in Fig. 4a, the four-step reaction proceeds to completion (100% yield of Ψ) and the conversion rate is even faster (∼5-fold) than in ΨMP synthesis. Lowered inhibition of the DeoB at decreased [phosphate] likely accounts for the observed rate acceleration. Interestingly, Ψ is released in the reaction to a concentration exceeding its solubility limit (200 mM) by ∼5-fold (Supplementary Table 1). Indeed, once the rearrangement of U was complete, Ψ is observed to crystallize spontaneously from the reaction mixture. Analysis of the crystalline powder revealed pure Ψ (≥ 99%). The reader will notice that in situ crystallization is one of the most desirable approaches of reaction product isolation, especially at larger scale in process chemistry [87][88][89] . It is interesting that Ψ shows much lower solubility (∼10-fold) than U. Preferred conformation in solution (Ψ syn; U anti) 90 might give an explanation. The anti conformation is expected to enable more extensive interactions with the water solvent than the syn conformation.
Contrary to ΨMP synthesis that involves phosphate as the substrate, the role of phosphate in Ψ synthesis is catalytic. The Ψ reaction thus required its own assessment of the [phosphate] to be used. Employing conditions otherwise as in Fig. 4a, the Ψ formation rate is shown to be dependent on the phosphate concentration (10-200 mM), with plateau (3.5 mM/min) reached at 100 mM (Supplementary Fig. 26a). The concentration of the intermediary ΨMP increases linearly with the [phosphate] used. At steady state during the reaction, the ΨMP accounts for ∼80% of the total phosphate present ( Supplementary Fig. 26b). The results imply limitation of the overall Ψ formation rate by the rate of dephosphorylation of ΨMP. Consequent variation of the Yjjg loading confirms the phosphatase as the ratedetermining enzyme, with a maximum Ψ formation rate of 5.8 mM/ min reached at 0.4 mg/mL (Supplementary Fig. 26a). Constancy of the Ψ formation rate at higher Yjjg loadings of up to 0.8 mg/mL suggests change in rate limitation to a different step, presumably that of DeoB. The portion of total phosphate bound in ΨMP reflects this change, by a 1.6-fold decrease to ∼50% (Supplementary Fig. 27b). The lowered [phosphate] in the reaction also reduced the Mn 2+ loading (2.5 mM) required to elicit the full enzyme activity (Supplementary Fig. 28a). Temperature variation reveals an optimum of the Ψ formation rate at 30°C ( Supplementary Fig. 28b), considerably lower than the temperature of maximum ΨMP formation rate (45°C; Supplementary  Fig. 8b). Sharp drop of the overall Ψ formation rate at elevated temperatures reflects slowdown of the conversion of ΨMP into Ψ and identifies Yjjg as the most thermolabile component of the enzyme cascade.
Using conditions updated for Ψ synthesis, we examined the U substrate concentration in the range 1.0-2.0 M. As already observed during ΨMP synthesis and even more strongly here, the Ψ formation rate decreases as the [U] is raised, suggesting a maximally usable substrate concentration of ∼1.2 M (Supplementary Fig. 29). There may well be biological (inhibition) and physical limits (viscosity) of the Ψ formation rate under the conditions used. Considering that the product solution obtained from conversion of 1.0 M U already reflects a 5-fold degree of oversaturation in Ψ, further reaction intensification to increase the product concentration seemed unnecessary. Variation in total enzyme loading at constant enzyme ratio reveals a linear scaling of the Ψ formation rate with the protein concentration (Supplementary Fig. 30). Enzyme usage and time needed to fulfill a given conversion task are thus reciprocally interrelated process variables of key importance for optimization. Simple engineering relationships are evidently crucial in order to cope with the inherent complexity of multienzyme systems in the development of biocatalysis applications 42,80,91 .

Scale up of Ψ production
The scale-up involved a 1000-fold change in reaction volume from 100 µL in the characterization to 100 mL in the production. An intermediate step at 10 mL was used additionally. Agitation was changed from revolution in the tube to stirring with a magnetic stirrer bar in the flask. The enzyme loading was minimized towards the aim of full conversion of U in 24 h. As shown in Supplementary Fig. 31, the Ψ release up to a concentration of ∼500 mM is identical within limits of error for the reactions done at different volume, indicating an entirely successful scale up. The reactions at 10 mL and 100 mL differ however from the 100 µL reaction in a much earlier onset of the crystallization of Ψ. Solid product starts to form already after ∼3 h when the U conversion is at ∼50%. Differences in vessel material (glass, plastic) and agitation mode used evidently result in strongly changed Ψ nucleation rates. However, the enzymatic rearrangement of the U proceeds unaffected by the piling up of solid material and an identical amount of Ψ is obtained from the unit volume in all reactions. Once the U is exhausted, the liquid-solid suspension is cooled (−20°C, 30 min) for the crystallization to complete. After lyophilization of the centrifuged solid, 24.3 g Ψ (white powder) is obtained in 95% isolated yield from the U used. The chemical structure of the Ψ is verified by 1 H, 13 [32][33][34][35][36][37][38] and the purity is shown as ≥99% by HPLC and assay for free phosphate.

Discussion
Biocatalytic process technology for the production of Ψ is presented. Domino reaction in four enzyme-catalyzed steps achieves a highly efficient N-C rearrangement of U. The cascade transformation of U into Ψ deserves, in our view, the designation "dream reaction" from a general organic synthesis as well as process chemistry point of view 48,[92][93][94][95] . It is atom-economic and selective in converting an expedient substrate (see below) in quantitative yield to the desired product. The reaction output exceeds by a large factor (≥ 5-fold) the thermodynamic boundaries of a stable product solution, enabling in situ crystallization of the Ψ for pure product isolation in ≥ 90% yield. The alternative (three-enzyme) cascade reaction to give ΨMP is similarly efficient by the criterions considered. Higher solubility of ΨMP than Ψ prohibits spontaneous crystallization. However, due to the excellent product purity (≥ 96%) received from the synthesis, a simple solvent removal is sufficient to isolate the ΨMP. Excluding steps required for the enzyme preparation, the production done at gram scale involves an extremely small E factor of just ∼3 (Ψ) and ∼1 (ΨMP) 38 . The waste is effectively water with phosphate, Mn 2+ and, unless recycled, enzymes remaining from the reaction. The process mass intensity (PMI) for the whole production (i.e., mass of all materials used, including the enzymes/mass Ψ or ΨMP isolated) is ∼4.5. Small-molecule processes in the pharma industry typically involve PMI values of several 10 to well over 100 38 . Similarly, the E factors are ≥ ∼25 38 . Compared to chemical syntheses of Ψ, the one-pot rearrangement of U into Ψ reported here offers fundamental improvements in process efficiency and sustainability, which both arise from the innovative cascade reaction development. Our considerations take into account recent developments in chemo-catalytic synthesis of C-nucleosides by List and co-workers 34 , even though the application to Ψ has not been shown in their study. Besides the core features of the synthetic transformation already discussed, the absence of an isolated intermediate in the multistep reaction sequence, the avoidance of excess reagents or organic solvents used, and the consistent usage of simple process operations are important engineering characteristics of the biocatalytic process.
Reporting the chemo-enzymatic synthesis of the U derivative Molnupiravir, McIntosh et al. 50 at Merck questioned the use of U as substrate, arguing that it could not be considered a "true commodity raw material" by the requirements of sustainable and green production. The U would have to be synthesized in several chemical steps from Rib and uracil. This led to the suggestion of an alternative Molnupiravir route that circumvents U entirely and starts from Rib and uracil instead. Disregarding the difficult debate on supply chain risks, we suggest that U produced efficiently by microbial fermentation [75][76][77] must also be brought into the picture. Full techno-economic analysis and life cycle assessment of possible Ψ process options will eventually be required to decide between U or Rib (plus uracil) as substrate used in manufacturing. From a technical process chemistry viewpoint, however, the rearrangement of U presents an interesting alternative. In principle, however, the YeiN reaction could be integrated with enzymatic reactions for Rib5P formation from Rib 73 . Ribose phosphorylation by ATP is an established reaction at scale 50,51 . As an additional note on substrate used, our four-enzyme cascade is flexible to also work with U-5'-monophosphate (UMP). Depending on market situation, the UMP may present a cost advantage over U. As shown in Supplementary  Fig. 39, release of phosphate from the UMP by Yjjg initiates the reaction sequence leading to N-C rearrangement into ΨMP and subsequent dephosphorylation to give Ψ. The UMP reaction differs from, and is less preferred from a sustainability point of view than, that of U in the larger (i.e., stoichiometric) amount of phosphate released into the product mixture.
Enzyme cascade transformations via phosphorylated or nucleotide-activated sugars enable innovative routes to complex molecules incorporating carbohydrate residue 41,96-100 . Applications are numerous in diversity-oriented synthesis at small scale 41,96,98,101,102 and interest for use in targeted production is rapidly growing 48,50,51,103 . However, telescoping multiple enzymatic reactions in one pot comes with significant challenges of process engineering (e.g., optimization) and control 38,42,99 . Here besides metrics of reaction efficiency already discussed, efficient use of enzyme catalyst is critical. Our g-scale synthesis of Ψ and ΨMP involves excellent protein mass-based turnover number (TONs) of 56 g/g and 129 g/g, respectively, received from just a single batch reaction without additional effort at enzyme recycling. On a mol basis, the individual enzyme TON is typically at ∼10 5 -10 6 (UP, Yjjg), somewhat smaller for YeiN and DeoB (∼10 4 ) due to the enzyme's lower specific activity. The TONs calculated from the experimental conversion data approach in magnitude the TTN values (Table 1) estimated for the individual enzymes based on separately determined rate constants for reaction and inactivation. The implications are twofold. First, the cascade reaction performed in batch process succeeds in exploiting almost the full catalytic potential of the enzymes used (Yjjg, YeiN, DeoB), as given by their respective TTN. Second, further improvement of the TON will require enzyme stabilization, by immobilization for example, to enhance the TTN. Additional point of importance is that simple (linear) scaling relationships between the production rate and the total protein concentration will facilitate process optimization under the constraints from economic assessment. The current productivities, 36 g/L/h for Ψ (Fig. 4b) and 30 g/L/h for ΨMP (Fig. 3c) push boundaries in general cascade biocatalysis, demonstrating how truly efficient such multienzyme systems can be. The TTNs mentioned above and the phosphate inhibition of DeoB represent immediate targets for further development through integrative approaches of enzyme development and process engineering.
Lastly, the conversion of U into Ψ and ΨMP embodies new chemistry realized by cascade biocatalysis. To our knowledge, multistep enzymatic N-C rearrangement has never been shown in a relevantly similar form. In natural product glycosides, glycosyltransferase-catalyzed two-step O-C rearrangement via sugar nucleotide intermediate was suggested for flavonoid C-glucoside synthesis in our earlier work 104 . In providing synthesis routes of high efficiency, as shown here for Ψ, the concept of glycoside rearrangement of naturally abundant substrates promotes the quest of new biocatalytic cascades, potentially facilitated by emerging tools of computer-aided synthesis planning 102 , and the search for the complementary set of enzyme activities.

Enzyme preparation
N-terminally His 6 -tagged YeiN, UP, DeoB, and Yjjg were used. All enzymes are encoded by E. coli genes. The relevant expression vector (pet15b _yein, pet15b _UP, pet15b_DeoB, and pet28a+_Yjjg) was transformed into E. coli BL21_DE3 (pLys) and selected on 0.1 mg/mL ampicillin LB-agar plates. Yjjg transformants were selected on 0.05 mg/ mL kanamycin LB-agar plates. Enzymes were expressed in 1-L baffled shaken flasks at 37°C and 110 rpm, using 250 mL LB -media containing 0.1 mg/mL of ampicillin. For Yjjg, expression was done at 0.05 mg/mL kanamycin. Cultures were inoculated to an OD 600 of 0.1. At an OD 600 of about 0.8, the temperature was decreased to 18°C and expression was induced with 0.4 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) for 20 h. Cells were harvested by centrifugation at 4420 × g at 4°C for 30 min using a Sorvall RC-5B refrigerated superspeed centrifuge (Du Pont Instruments, Newtown, CT, USA). The supernatant was discarded and the pellet was suspended in His-tag binding buffer (50 mM HEPES, pH 8.0 containing 500 mM NaCl, 30 mM imidazole, 5% glycerol). Cells were disrupted using sonication (Fisherbrand Sonic Dismembrator, model Ultrasonic Processor FB-505; Fisher Scientific, Vienna, Austria; 8 min) on ice and the cell-free supernatant was recovered by centrifugation at 4°C and 27,150 × g for 50 min. Pre-treated cell lysate (20 mL) was loaded on 2 × 5 mL HisTrap FF column (Cytiva, Marlborough, MA, USA), equilibrated with His-tag binding buffer, and mounted on an ÄKTA prime plus (Cytiva) system. Protein purification was performed at 10°C and flow rate of 3 mL/min. Protein was eluted using an imidazole gradient from 0% to 100% His-tag elution buffer (50 mM HEPES, pH 8.0, containing 500 mM NaCl and 300 mM imidazole). Fractions containing the target protein were pooled, concentrated, and buffer-exchanged with an Amicon Ultra-15 Centrifugal Filter Units (Millipore; Billerica, MA, USA). The final protein concentration was 30-150 mg/mL in 50 mM HEPES buffer containing 5% glycerol (v/v), 500 mM NaCl and 2 mM MnCl 2 (pH 7.0). Enzyme was stored at −20°C until further use. Protein purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Supplementary Fig. 1).

Initial rate analysis
This was used for standard enzyme activity measurement and determination of kinetic parameters. A 50 mM HEPES buffer (pH 7.0) containing 2.0 mM MnCl 2 was used at 30°C. Standard activities were also recorded at 40°C. Incubations were done in a total volume of 200 µL using an Eppendorf (Hamburg, Germany) thermomixer with gentle agitation (300 rpm). The substrate concentrations used are specified below for each enzyme. Reactions were started by adding concentrated enzyme solution (≤2% of total volume) to the temperatureequilibrated substrate solution. Activity determinations were performed at conditions of substrate saturation. Kinetic parameter determination involved initial rates measured at variable substrate concentrations (≥6 concentrations). In two-substrate reactions (UP, YeiN), the concentration of the second substrate was constant and saturating. Samples (5 µL) were taken at certain times and unless mentioned otherwise, quenched in methanol:water 1:1 (v:v; 100 µL), centrifuged, and analyzed by HPLC. Initial rates were acquired from the linear range of product formed (or substrate consumed) with time under conditions of ≤20% conversion of the substrate(s) used. One unit (U) is the enzyme amount for 1 µmol/min of product release or substrate consumed under the assay conditions used. Kinetic parameters (V max , K M ) were obtained from non-linear least squares fit of Eq. (1) to the initial-rate data. Fitting was done with SigmaPlot V10.0 (Systat, Erkrath, Germany). In Eq. (1), V is the initial rate dependent on [S], V max is the maximum initial rate (µM/min), K M is the Michaelis constant (mM) and [S] is the initial substrate concentration (mM). The specific activity was determined from the relationship V spec = V max /[E], where [E] is the enzyme concentration.
[E] was determined from the protein concentration, measured by 280 nm absorbance, and calculated with the protein-specific molar extinction coefficient.
The individual enzyme reactions were done as described below. DeoB. Enzyme-coupled assay was developed to measure the activity of DeoB. Initial rates were recorded in the presence of 10 mM uracil and 1.5 mg/mL YeiN. The DeoB was used at 0.0013 mg/mL. Under the conditions used, the DeoB activity is completely rate limiting and the ΨMP formation rate, therefore, equals the Rib1P isomerization rate. Standard-specific activity was measured at 3.0 mM Rib1P. For kinetic parameter determination, Rib1P was varied in the range 0.01-1.00 mM.
To characterize the enzyme inhibition by phosphate, initial rates were measured at three different phosphate concentrations in the range 0.5-1.5 mM. The Rib1P concentration was varied in the range 0.03-0.6 mM. The inhibition was found to be noncompetitive, decreasing the V max while leaving the K M unaffected. The data were fitted with Eq. (2) where K I is the inhibition constant and [I] is the inhibitor concentration.
Activities (U) and other rates refer to ΨMP (=Rib5P) released. Operational DeoB activity in the three-enzyme cascade was obtained at 40°C in 1.0 M potassium phosphate buffer (pH 7.0) containing 20 mM MnCl 2 and 1.00 M U. The reaction was started by adding 0.25 mg/mL UP, 1.5 mg/mL YeiN and DeoB in the strictly limiting concentration of 2.5 mg/mL. The operational activity in the four-enzyme cascade was obtained at 30°C in 1.0 M potassium phosphate buffer (pH 7.0) containing 2.5 mM MnCl 2 and 1 M U. The reaction was started by adding 0.5 mg/mL UP, 1.5 mg/mL YeiN, 0.2 mg/mL Yjjg and DeoB in the strictly limiting concentration of 2.5 mg/mL. Retention of DeoB activity (∼10% of basal activity) at the high phosphate concentrations used in the "operational" assays suggests that the inhibition by phosphate is only partial, with residual activity in the phosphate-saturated enzyme.
Yjjg. ΨMP or Rib5P was used as the substrate. The reaction was started by adding 0.0066 mg/mL (ΨMP) or 1.0 mg/mL (Rib5P) of enzyme. Samples (5 µL) were heat treated (95°C, 5 min) and analyzed by HPLC (ΨMP reaction) or with a colorimetric phosphate assay 105 (Rib5P reaction). Standard-specific activities were measured at 40 mM ΨMP. For kinetic parameter determination, the concentration of ΨMP or Rib5P has varied in the range 1.0-30 mM. Activities (U) and other rates refer to Ψ or phosphate released.
Thermal shift assay for melting temperature determination Differential scanning fluorometry was used. Aliquots (45 µL) of enzyme solution (5 µM) in 0.10 M or 1.0 M potassium phosphate buffer (pH 7.0) were mixed with 5 µL of 200 × SYPRO orange solution and aliquoted in triplicate into a 96-well-PCR plate (Bio-Rad, Hercules, CA, USA). The plates were sealed with optical-quality sealing tape (Bio-Rad) and heated in a Bio-Rad CFX Connect Real-Time PCR Detection System, from 25°C to 99°C in increments of 0.5°C and hold 30 sec at each step. The excitation and emission wavelengths were 490 and 575 nm, respectively, and the fluorescence changes in the wells were monitored continuously. CFX Maestro (Bio-Rad; Version 4.0.2325.0418) was used for data processing to calculate the melting temperature T m . Buffer without enzyme was the control.
To analyze the reaction mixture of conversion of ΨMP to ΨTP, the HPLC method was adapted slightly. A Kinetex C18 column (Phenomenex; 5 μm, 100 Å, 50 × 4.6 mm) was used. The eluting buffer from above additionally contained 12.5% (by volume) acetonitrile. A 5-min long isocratic run at 2.0 mL/min flow rate was used. Under these conditions, the retention times were as follows (Supplementary

Thin-layer chromatography (TLC)
About 1-2 μL sample were spotted onto a TLC plate (Merck, Darmstadt, Germany). The mobile phase was composed of 2-BuOH:AcOH:H 2 O (2:1:1). The development solution consisted of 0.5 g thymol, 95 mL EtOH and 5 mL H 2 SO 4 . Spots were developed by heating the plate to~70°C for 2 min using a heat gun.

NMR analysis of purified reaction products
Lyophilized products were dissolved to 5.00−100 mM in D 2 O in a total volume of 600 μL and transferred to a 5 mm high-precision NMR sample tube. Samples were analyzed on a Varian INOVA 500-MHz NMR spectrometer (Agilent Technologies, Santa Clara, California, USA) using the VNMRJ 2.2D software for the measurements, (Agilent Technologies, Santa Clara, CA, USA) or on a Bruker AVANCE III 300-MHz spectrometer (Bruker, Rheinstetten, Germany) with an autosampler and the Bruker Topspin 3.5 software for measurements.
Reaction parameter variation for ΨMP synthesis using the three-enzyme cascade The standard (reference) conditions on which to perform singleparameter variation were 1.5 M potassium phosphate buffer (pH 7.0), 1.0 M U, 10 mM MnCl 2 , 1.0 mg/mL UP, 10 mg/mL DeoB and 6.0 mg/mL YeiN. Reactions were performed in 100 µL scale and incubated at 30°C. The pH was varied in the range 6.5-8.0 in 0.5 pH increments. [phosphate] was varied from 1.0 M to 1.5 M in 0.1 M increments. Temperature was varied in the range 30-50°C in 5°C increments. Addition of Glc1,6diP was tested at 100 µM.

Enzyme stability studies
Enzymes were incubated under conditions mimicking those of the cascade reactions to examine the stability of their activities. The conditions used were 1.0 M potassium phosphate buffer (pH 7.0) containing 20 mM MnCl 2 at 40°C (three-enzyme cascade) and 0.1 M potassium phosphate buffer (pH 7.0) containing 2.5 mM MnCl 2 at 30°C (four-enzyme cascade). The enzyme concentrations used in the incubations were as follows: UP, 0.3 mg/mL; DeoB, 4.0 mg/mL; YeiN, 2.1 mg/mL (three-enzyme cascade); UP, 0.3 mg/mL; DeoB, 4.0 mg/mL; YeiN, 3.0 mg/mL; Yjjg 0.1 mg/mL (four-enzyme cascade). Residual enzyme activity was measured in samples after 4 h and 16 h, using the enzyme-specific assays described above. Data were plotted in semilogarithmic form (activity vs. time; Supplementary Fig. 4) and the approximate slope was used to estimate an apparent first-order inactivation rate constant (k d ). The total turnover number (TTN) of enzyme is given by Eq. (3) where k cat is the apparent catalytic constant which is calculated from the specific enzyme activity under these conditions (U/mg) and the enzyme molecular mass (g mol −1 ). Note that the TTN thus calculated is based on mol.
Synthesis of 1 g ΨMP The reaction was performed at 5 mL total volume in a 15 mL Sarstedt tube (Biedermannsdorf, Germany). Incubation was at 40°C in a shaking water bath 1083 (GFL, Burgwedel, Germany

ΨMP isolation
Enzymes were removed from 5 mL (∼1 M ΨMP) reaction mixture with an Amicon Ultra-15 Centrifugal Filter Unit (Millipore; Billerica, MA, USA) of 10 kDa molecular mass cut-off. The supernatant was lyophilized using a Christ Alpha 1-4 freeze drier (bbi-biotech GmbH, Berlin, Germany) attached to a Vacuubrand vaccum pump unit RZ 6. The solid recovered is ΨMP in technical-grade purity. The product was analyzed by HPLC and NMR, and phosphate was measured using a colorimetric assay 105 . The isolated yield reported is based on the mass calculated from the UV-absorbance of the isolated ΨMP. Prior to 31 P-NMR measurements Mn 2+ was removed by treatment with Amberlite® IRC120 H.

Ψ isolation
The reaction mixture from the Ψ synthesis (20 to 100 mL, ∼1 M Ψ) was cooled to −20°C for 30 min to maximize Ψ precipitation. The supernatant was separated from precipitated Ψ using centrifugation at 2880 × g and 4°C for 10 min. The precipitated Ψ was lyophilized using instrument described above. The solid recovered is Ψ in high purity. The product was analyzed by HPLC and NMR.
Water solubility of Ψ, U, and uracil Solubility was examined in the range 30-50°C in increments of 5°C. The solid compound (Ψ, U, or uracil) was added in steps (1 mg) to Milli-Q water (1.0 mL) and incubated under agitation at 1200 rpm (Eppendorf thermomixer) for 30 min. Undissolved material was removed by centrifugation at 27,150 × g for 5 min with an Eppendorf 5415 R microcentrifuge. The nucleoside and nucleobase concentration in solution was measured by 260 nm absorbance and calculated with the respective molar extinction coefficient, as described under "Determination of nucleotide and protein concentration".

YeiN catalyzed conversion of 100 mM uracil
The reaction was performed in 1.0 mL total volume using a 2 mL glass vial placed in a water bath. Temperature and stirring were controlled using a magnet stirrer (IKA 3581200, Staufen, Germany) with integrated heating plate. The substrate solution contained 100 mM uracil and 150 mM Rib5P in water supplemented with 10 mM MnCl 2 . The pH was set to pH 7.0 using 1 M NaOH. The reaction was started with 0.2 mg/mL YeiN and the incubation was continued at 37°C and 900 rpm agitation rate. Samples were taken at certain times. They were centrifuged at 4°C and 27,150 × g for 1 min to remove insoluble uracil. The combined Ψ and uracil concentrations were determined photometrically, and the relative content was analyzed by HPLC, allowing for the determination of the absolute Ψ and uracil concentrations.

Synthesis and isolation of ΨTP
The synthesis was performed in 10 mL total volume using a 15-mL Sarstedt tube for incubation at 37°C in a water bath. The solution 50 mM HEPES buffer (pH 8.0) contained 20 mM ΨMP, 5.0 mM ATP, 60 mM phosphoenolpyruvate, 2.0 mM MgCl 2 , 1.5 mg/mL cytosine 5′monophosphate kinase 73 and 0.2 mg/mL pyruvate kinase (PK; Sigma-Aldrich, Darmstadt, Germany). The incubation was performed for 20 h. Suitable conversion of the ΨMP substrate (>90%) was shown by HPLC. Enzymes were removed with an Amicon Ultra-15 Centrifugal Filter Unit (Millipore; Billerica, MA, USA) of 10 kDa molecular mass cut-off. The filtrate was loaded onto a XK26/40 column packed with 125 mL DEAE FF anion exchange resin (both Cytiva) and mounted onto an ÄKTA prime plus FPLC system (GE Healthcare; Chicago, IL, USA). The column was equilibrated in 5 mM NH 4 HCO 3 solution (pH 8.0). Elution was done at a flow rate of 10 mL/min using 200 mM NH 4 HCO 3 . Products were eluted at 80-100 mM NH 4 HCO 3 . Elution was monitored at 260 nm. Fractions containing ΨTP were pooled and concentrated using a Laborta 500-efficient rotary evaporator (Heidolph Instruments, Schwabach, Germany) operated at 40°C and 30 mbar. The concentration was done until all NH 4 HCO 3 was evaporated. Finally, the samples were lyophilized using a Christ Alpha 1-4 freeze drier (bbibiotech GmbH, Berlin, Germany) using a Vacuubrand vacuum pump unit RZ 6. The isolated product was analyzed by HPLC as well as 1 H, 13 C, and 31 P NMR ( Supplementary Figs. 22-25).

Synthesis and isolation of Rib1P
The synthesis was performed in 500 µL total volume in 1.4 M potassium phosphate buffer (pH 7.0) containing 2.0 M U. The reaction was started with 11 mg/mL UP and the incubation was continued at 40°C and 350 rpm agitation rate overnight. Precipitated Ura was removed by centrifugation at 4°C and 27,150 × g for 10 min, followed by enzyme removal using an Amicon Ultra-15 Centrifugal Filter Unit (Millipore) with a molecular mass cut-off of 10 kDa. Phosphate was precipitated using barium acetate (mol equivalent of the phosphate present, used as crystalline powder) and the formed solids were removed by centrifugation at 4°C and 27,150 × g for 10 min. The supernatant was loaded onto a XK16/20 column packed with 25 mL DEAE FF anion exchange resin (both Cytiva) and mounted on an ÄKTA prime plus FPLC system (GE Healthcare). The column was equilibrated in 5 mM NH 4 HCO 3 (pH 8.0). Rib1P was eluted using a linear gradient from 0 to 100% of 100 mM NH 4 HCO 3 in 250 mL with a flow rate of 8 mL/min. Fractions were monitored for Rib1P (TLC) and phosphate content (colorimetric assay 105 ). Fractions containing Rib1P and showing no detectable phosphate content were pooled and concentrated to a final volume of 50 mL using a Laborta 500-efficient rotary evaporator (Heidolph Instruments) operated at 40°C and 30 mbar. The concentration was repeated as necessary until all NH 4 HCO 3 was evaporated. The Rib1P concentration of the stock solution was determined from the phosphate released upon incubation with calf intestine phosphatase. The chemical structure and identity of the isolated compound as Rib1P were confirmed by 1 H NMR (Supplementary Fig. 42).

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
All data reported in the paper are available from the corresponding author (B.N.) upon request. Additionally, source data are provided in this paper for Figs